EP1131664A1 - Verfahren zur einstellung der systemparameter eines laserscanmikroskops - Google Patents
Verfahren zur einstellung der systemparameter eines laserscanmikroskopsInfo
- Publication number
- EP1131664A1 EP1131664A1 EP99962046A EP99962046A EP1131664A1 EP 1131664 A1 EP1131664 A1 EP 1131664A1 EP 99962046 A EP99962046 A EP 99962046A EP 99962046 A EP99962046 A EP 99962046A EP 1131664 A1 EP1131664 A1 EP 1131664A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- dialog
- system parameters
- resolution
- proposed
- user
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
Definitions
- the problem situations could be different problem cases, for example the problem case according to which the sample bleaches too strongly (in the case of fluorescent objects), according to which the image data are too "noisy", after which the measuring time is too long or after which the resolution of the image recording increases
- the dialog offers assistance or solutions that can be selected by the user on the basis of preset system parameters, and on the basis of the assistance provided in the dialog, optimization - in dialog - is possible.
- system parameters suggest mutually exclusive properties or settings in the dialog and are automatically set after selection and, if necessary, after confirmation. These mutually influencing or even excluding properties are shown in FIG. 1 in the sense of a "quality triangle".
- the mutually exclusive or counteracting factors are the maximum resolution, maximum brightness and maximum recording speed.
- system parameters are retrieved from an expert system stored in the database, taking into account the specifications, this expert system on the one hand
- Empirical values and on the other hand algorithms can take into account the quality triangle. Furthermore, it is conceivable that the system parameters taking into account the specifications using Fuzzy logic is determined and set after selection or automatically - if necessary after confirmation by the user - measures according to fuzzy logic can be incorporated into the expert system.
- the recording or system parameters set or changed by the user could be analyzed by the recording program. If one or more system parameters were set “wrong" by the user, the program can automatically and on its own initiative lead the user into a dialog with the aim of restoring an optimal system setting. To do this, a "quality demon module" of the program would have to Use the current system parameters continuously and use the dialog module automatically if the setting is not optimal.
- the user guidance could lead a learning program in dialogue, according to which the user is guided to optimal recording strategies, on the basis of object or problem-specific system settings.
- the interactive learning program could also be used as a Training program designed for experienced users, namely, to be able to increase the quality of work of users working with the fluorescence microscope.
- the user is provided with user guidance or a computer dialog, according to which, depending on the specific application, an optimal system setting is possible and corresponding recording strategies are proposed and can be selected by the user.
- an optimal system setting is possible and corresponding recording strategies are proposed and can be selected by the user.
- the "quality triangle" shown in FIG. 1 can be included in the optimization.
- the method according to the invention provides the user with tools that enable him, in the form of computer-aided user guidance, for all conceivable problems, in particular for the four aforementioned most common problems. Receive suggestions for solutions, also based on selectable presets. If an expert system is incorporated in the user guidance, the user can fall back on empirical values from various microscopic applications and questions. It is again conceivable to implement a system that is capable of learning, ie a file that expands with each positive image acquisition - automatically or after the user has expressly confirmed it. Methods such as "fuzzy logic" can be implemented, from which the system settings can be further optimized.
- Focus area and the optimal irradiance in focus Focus area and the optimal irradiance in focus.
- the maximum imaging scale of the lens should not exceed 50 (10 mm / 200 ⁇ m) and its working distance should be in the order of the sample thickness (0.1 mm).
- the maximum numerical aperture (NA) should be selected under these secondary conditions.
- the lateral resolution of the microscope is between 210 nm and 320 nm with the selected objective and taking into account the specified emission wavelength of the fluorescence molecules. The exact value depends on the set pinhole radius. Taking into account the Nyquist theorem, the number of pixels /? To be recorded per line is:
- the number of pixels in the image can be set to the values 256 2 , 512 2 or 1024 2 .
- the maximum number of pixels is 1024 2 . Since this number is below the values calculated according to Nyquist, artifacts may occur in the image. If this is the case, either the scan field must be reduced using the zoom, or a lens with a smaller numerical aperture must be used.
- the optimal irradiance is calculated as follows:
- E ⁇ «9.5 - 10 ⁇ ⁇ ( ⁇ s + W ⁇ ⁇ r ⁇ ) cm" - s
- the optimum irradiance E s is greater by a factor of 8 in the concrete example. The neglect is generally justified if the detection time per pixel is less than the lifetime of the triplet state. If the detection time is of the order of the life of the
- Triplet state as in the present example, their existence should be taken into account when calculating the optimal irradiance.
- the laser power must be set to approx. 1 mW.
- the signal-to-noise ratio should not fall below a certain threshold.
- the microscope With a set detection pinhole radius of approx. 2 optical units, the microscope almost reaches the maximum axial resolving power.
- the lateral resolution is approx. 260 nm.
- the detected pixel volume Vp ixe ⁇ is obtained from the axial resolution and the lateral resolution assuming an ellipsoidal focus:
- the maximum density p of the fluorescence molecules is 200 molecules per ⁇ m 3, so the number of molecules in focus is ⁇ / focus
- the emission rate E m is equal to the excitation rate.This means that half of the molecules are in the basic state and the other in the excited state in dynamic equilibrium. In the present example, triplet states are not negligible. The emission rate per molecule is therefore
- the fading of the sample has the effect that the brightness of the last recorded layers is approximately 80% of the first recorded layers.
- the signal-to-noise ratio is reduced to approx. 2.
- the photomultiplier voltage needs to be adjusted to the maximum signal height of approximately 5 photons per pixel to be expected. For the given recording time per pixel, the photomultiplier voltage is approx. 720 V.
- the lifespan of the triplet state can be reduced by the presence of, for example, oxygen, or, in the case of many dyes, is already lower than that of the fluorescein triplet state in the isolated state.
- a shortening of the lifetime of the triplet states causes an increase in the optimal irradiance, higher emission rates and thus also a better signal-to-noise ratio in the image.
- the sample is more bleached during exposure due to the higher radiation exposure.
- the range of bleaching rates for the different dyes is very broad - depending on the environment of the molecules - and can be reduced many times over for solid preparations using anti-bleaching agents.
- the bleaching rate of fluorescein is a typical mean value.
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19853407 | 1998-11-19 | ||
DE1998153407 DE19853407C2 (de) | 1998-11-19 | 1998-11-19 | Verfahren zur Einstellung der Systemparameter eines konfokalen Laserscanmikroskops |
PCT/DE1999/003527 WO2000031576A1 (de) | 1998-11-19 | 1999-11-05 | Verfahren zur einstellung der systemparameter eines laserscanmikroskops |
Publications (2)
Publication Number | Publication Date |
---|---|
EP1131664A1 true EP1131664A1 (de) | 2001-09-12 |
EP1131664B1 EP1131664B1 (de) | 2003-07-02 |
Family
ID=7888361
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19990962046 Expired - Lifetime EP1131664B1 (de) | 1998-11-19 | 1999-11-05 | Verfahren zur einstellung der systemparameter eines laserscanmikroskops |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1131664B1 (de) |
JP (1) | JP4139566B2 (de) |
AT (1) | ATE244414T1 (de) |
DE (2) | DE19853407C2 (de) |
WO (1) | WO2000031576A1 (de) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9348127B2 (en) | 2008-09-30 | 2016-05-24 | Carl Zeiss Microscopy Gmbh | Methods and apparatuses for structured illumination microscopy |
Families Citing this family (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6859273B2 (en) | 2001-07-23 | 2005-02-22 | University Of Rochester | Method for operating a laser scanning confocal microscope system and a system thereof |
DE10150542B4 (de) * | 2001-10-12 | 2007-03-29 | Leica Microsystems Cms Gmbh | Verfahren zur Fluoreszenzmikroskopie |
DE10161613A1 (de) * | 2001-12-15 | 2003-06-18 | Leica Microsystems | Verfahren zur Selbstüberwachung eines Mikroskopsystems, Mikroskopsystem und Software zur Selbstüberwachung |
DE10229407B4 (de) | 2002-06-29 | 2021-10-14 | Leica Microsystems Cms Gmbh | Verfahren zur Einstellung der Systemparameter eines Rastermikroskops und Rastermikroskop |
DE10324329B4 (de) * | 2003-05-29 | 2008-05-15 | Leica Microsystems Cms Gmbh | Objektträgervorrichtung zur Untersuchung mit einem Mikroskop |
DE10361158B4 (de) * | 2003-12-22 | 2007-05-16 | Leica Microsystems | Einrichtung und Verfahren zur Konfiguration eines Mikroskops |
DE102004031570B4 (de) | 2004-06-12 | 2008-04-10 | Leica Microsystems Cms Gmbh | Objektträgervorrichtung zur Untersuchung mit einem Mikroskop |
DE102004064072B4 (de) | 2004-06-12 | 2020-06-10 | Leica Microsystems Cms Gmbh | Objektträgervorrichtung zur Aufnahme eines mit einem Mikroskop zu untersuchenden oder mit einem Laboranalysesystem zu analysierenden Objekts sowie Verfahren |
EP1669740A1 (de) * | 2004-12-10 | 2006-06-14 | Olympus Corporation | Mikroskopgerät, Sensitivitätseinstellungsverfahren für einen Photodetektor, Steuereinheit und Speichermedium |
DE102007007395A1 (de) * | 2007-02-12 | 2008-08-14 | Leica Microsystems Cms Gmbh | Mikroskop |
DE102007061215B4 (de) * | 2007-12-19 | 2023-01-12 | Carl Zeiss Microscopy Gmbh | Verfahren zur automatischen Anpassung einer Mikroskopbeleuchtung an Filter |
DE102008016262B4 (de) | 2008-03-29 | 2016-05-04 | Carl Zeiss Microscopy Gmbh | Verfahren zur Ermittlung der Konfiguration eines Mikroskops |
JP2014523545A (ja) * | 2011-06-30 | 2014-09-11 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | 生体イメージングのための顕微鏡システム及び方法 |
WO2013002720A1 (en) * | 2011-06-30 | 2013-01-03 | Ge Healthcare Bio-Sciences Corp | Image quality optimization of biological imaging |
DE102018132337A1 (de) * | 2018-12-14 | 2020-06-18 | Leica Microsystems Cms Gmbh | Mikroskopsystem mit Eingabeeinheit zum gleichzeitigen Einstellen von wenigstens drei Einstellparametern mittels eines in einer Eingabefläche positionierbaren Eingabezeigers |
US20230176355A1 (en) * | 2020-06-08 | 2023-06-08 | Leica Instruments (Singapore) Pte. Ltd. | Microscope System and Corresponding System, Method and Computer Program for a Microscope System |
DE102020115610A1 (de) | 2020-06-12 | 2021-12-16 | Leica Microsystems Cms Gmbh | Verfahren, Recheneinheit und System zum Bestimmen jeweils eines Werts für wenigstens drei Einstellparameter mittels einer Eingabeeinheit in Form einer graphischen Benutzer-Schnittstelle |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE2929545A1 (de) * | 1979-07-20 | 1981-01-29 | Heidenhain Gmbh Dr Johannes | Verfahren und schaltungsanordnung zur programmerstellung und/oder programmaenderung bei numerisch gesteuerten maschinen |
DE3933064C2 (de) * | 1988-10-05 | 1994-02-10 | Olympus Optical Co | Steuervorrichtung für ein Mikroskop |
FR2652928B1 (fr) * | 1989-10-05 | 1994-07-29 | Diadix Sa | Systeme interactif d'intervention locale a l'interieur d'une zone d'une structure non homogene. |
US5483055A (en) * | 1994-01-18 | 1996-01-09 | Thompson; Timothy V. | Method and apparatus for performing an automatic focus operation for a microscope |
US5923466A (en) * | 1993-10-20 | 1999-07-13 | Biophysica Technologies, Inc. | Light modulated confocal optical instruments and method |
JPH09511579A (ja) * | 1994-04-11 | 1997-11-18 | ライカ アーゲー | 測定点の位置データを求める方法および光路の倍率測定装置 |
DE19654211C2 (de) * | 1996-12-24 | 2000-07-06 | Leica Microsystems | Konfokales Mikroskop |
JPH11183806A (ja) * | 1997-12-18 | 1999-07-09 | Nikon Corp | コンフォーカル顕微鏡 |
-
1998
- 1998-11-19 DE DE1998153407 patent/DE19853407C2/de not_active Expired - Fee Related
-
1999
- 1999-11-05 DE DE59906211T patent/DE59906211D1/de not_active Expired - Lifetime
- 1999-11-05 JP JP2000584335A patent/JP4139566B2/ja not_active Expired - Lifetime
- 1999-11-05 EP EP19990962046 patent/EP1131664B1/de not_active Expired - Lifetime
- 1999-11-05 AT AT99962046T patent/ATE244414T1/de active
- 1999-11-05 WO PCT/DE1999/003527 patent/WO2000031576A1/de active IP Right Grant
Non-Patent Citations (1)
Title |
---|
See references of WO0031576A1 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9348127B2 (en) | 2008-09-30 | 2016-05-24 | Carl Zeiss Microscopy Gmbh | Methods and apparatuses for structured illumination microscopy |
Also Published As
Publication number | Publication date |
---|---|
DE19853407C2 (de) | 2003-09-11 |
DE59906211D1 (de) | 2003-08-14 |
JP4139566B2 (ja) | 2008-08-27 |
ATE244414T1 (de) | 2003-07-15 |
EP1131664B1 (de) | 2003-07-02 |
DE19853407A1 (de) | 2000-05-31 |
WO2000031576A1 (de) | 2000-06-02 |
JP2002530715A (ja) | 2002-09-17 |
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